The focus of our research is to accelerate the understanding of basic mechanisms involved in genetics via the study by exploiting the data emerging from the human genome project and the genome of other species. This spans the range from de novo genomic sequencing, to re-sequencing, genotyping, epi-genotyping, bulk cytogenetic and histologic inspection, phenotype measurements. The overall intent is to expand our knowledge and understanding, to improve medicine and to improve the conditions of humans and other species on the planet earth. The developments in our lab are applied and validated by investigating cancer, cardiac disease (the number 1 and number 2 killers of Americans) and other diseases such as inflammation, infection and fertility. Frequently our work is done in collaboration with various esteemed researchers at UTSW and elsewhere. Of particular note is our work with the cancer center and NIH SPORE in lung cancer at UTSW as well as with numerous physician researchers in heart disease, diabetes and dermatology.
Two particular areas of focus on the global study of known or predicted hot-spots for polymorphism in the genome that can play important mechanistic roles in genetics via protein sequence or splicing alteration, expression modulation via promoter alterations, epi-genetic alterations such a adherent methylation, and changes in protein-DNA binding properties.
Areas of Emphasis in Genomics/Porteomics
Global directed search for polymorphisms Cancer Research Cardiac Disease Research Light Biology Patterning device and associated chemistry for tissue engineering
Global directed search for polymorphisms - Using our predictive codes for simple sequence repeat polymorphisms (Pompous, Rep-X) and single nucleotide polymorphisms (SNIDE), we have created catalogues of highly-probable variants. These catalogues, which contain gene entries that span all of medicine and biology, are a source for a directed search for genetic variation that are causative of disease. We have been able to exploit these techniques to make substantive statements in genetics, evolution and quantitative traits. For example, we have shown, using dogs as a model, while re-sequencing ~30 genetic coding regions of ~200 canines of ~100 breeds, primarily in transcription factors, that the microsatellites are significantly polymorphic, while there are very few SNPs that can account for the diversification of the species into the abundant number of breeds that have emerged very quickly, i.e. in the last 1,000 years or so. We further demonstrated that quantitative traits (nose length, etc.) in genes previously implicated in cranial-facial abnormalities were highly correlated with the quantitative expansion of the microsatellites. In a separate study, we have also shown that SNPs are non-randomly distributed in the genome and in a gene. In particular, in SNPs that have been shown to be causative of phenotype and disease via entry in the Human Gene Mutation Database are very non-random, where for example, an ARG to stop transition appears >100 more frequently than expected. We have been able to demonstrate that we can decouple the mutation rate and the response to selection pressure.
Epi-genetic analysis using microarrays – We have developed techniques for the global measurement of chromatin status (DNA packing) on a resolution of a single gene. This was done by merging techniques for the enrichment of DNA from cell lines for the packed fraction and then using cDNA arrays to readout differential packing status of different cell lines or with different treatments. This data correlates well with gene expression data, but is revealing of the mechanism by which expression is modulated, and it was interesting to find that there are certain genes that escape strong packing and are expressed at high levels. This could lead to a new technique for cancer drug discovery or evalutation.
Gene Expression Analysis - We have been developing new protocols for improved standardization of spotted and other microarrays. This has included the development of genomic labeling (where DNA is used as the standard) and with RNA pools developed by Stratagene.
Cancer Research - In close collaboration with John Minna, M.D., our group has been developing and then applying our genomic sequence analysis tools, especially Panorama and Pompous, to study lung cancer. With John, we have analyzed in depth human chromosome 3p for tumor suppressor genes and genetic variations that contribute to cancer progression. This research is expanding to incorporate identification of methylation and regulatory control elements of cancer genes in software and then laboratory analysis on expression microarrays and re-sequencing (DOC) arrays. An example: With John Minna, the following demonstrated for one of our predicted simple sequence repeats that it was polymorphic and was informative in that it could differentiate different patients and the state of the lung cancer tumor (LOH was observed in some patients tumor DNA compared to their normal cell DNA).
Cardiac Disease Research - We are working with several groups at UTSW to understand the genetics of heart disease. To identify the genes that contribute to heart disease and risk, we are identifying candidate genes using experts, microarray analysis of mouse models, and our computer codes (Arrogant, ELXR, text data mining). At least 600 genes identified were completely re-sequenced in substantial sets of cohorts to identify polymorphisms, providing the basis of subsequent mass spec-based assays will be developed by Sequenom and SNP analysis using Perlegen microarrays to then genotype 3,500 patients collected by the Reynold's Center.
de novo DNA Sequencing - In collaboration with Ward Wakeland, Ph.D., we have completed shotgun sequencing a tiling path of BACs that span a region of the mouse genome involved in inflammatory disease. This contiguous region is 941 kilobases in length and analysis using our computer codes has indicated that it is gene rich. We used the Beckman CEQ capillary sequencers to read the shotgun double-stranded plasmid fragments, which were then assembled using phred/phrap/consed running on our HP and other UNIX servers.
Sequencing and Re-sequencing of difficult regions - We have been developing protocols for capillary-based sequencing, specializing in problematic sequence regions. This has included some regions not previously sequenced that included secondary structure that prevented easy inclusion of dye terminators (this has been reported in Biotechniques) and the sequencing of simple sequence repeat polymorphisms that by their vary nature are difficult to sequence, especially for heterozygotes. We have rugged protocols for PCR amplification of these regions and for sequencing using the unique Infra-red chemistry used in the Beckman CEQ sequencers.
Light Biology - In the area we now call light biology, we have been investigating new biomedical applications of light projection and detection. In the area of light projection using Texas Instruments Digital Light Processing chips (DLP), this has included the development of the DOC system for chip-based re-sequencing and expression analysis, modulation of the immune system using specialized light sources (VSS), tissue engineering. This formed the basis for a company, later sold to Nimblegen, Inc (now Roche). We have now constructed a peptide/peptoid custom microarray synthesis technology based on our initial work manufacturing oligonucleotide microarrays. In the area of light detection, we have focused on developing cytogenetics, re-sequencing and expression methods that use hyperspectral imaging to enable more accurate determination of labeled probes and a higher depth of probe multiplexing.
Patterning device and associated chemistry for tissue engineering-We have devised a cell patterning technology that can be extended three dimensionally to direct the growth and development of multi-cellular tissue structures. The technology couples light activated growth factors with high precision light projection of UV light using the DLP technology as well.
